Submitted on March 14, 2008
Revised on June 5, 2008
Accepted on June 9, 2008
Factors Released from Embryonic Stem Cells inhibit Apoptosis in H9c2 cells through P1-3kinase/Akt but not ERK pathway
Dinender K Singla1*, Reetu D Singla1, and Debbie E McDonald2
1 University of Central Florida
2 University of Vermont
* To whom correspondence should be addressed. E-mail: dsingla{at}mail.ucf.edu.
We recently reported that embryonic stem cells-conditioned medium (ES-CM) contains antiapoptotic factors that inhibit apoptosis in the cardiac myoblast, H9c2 cells. However, the mechanisms of inhibited apoptosis remain elusive. In this report, we provide evidences for novel mechanisms involved in the inhibition of apoptosis provided by ES-CM. ES-CM from mouse ES cells was generated. Apoptosis was induced after exposure with H2O2 (400µm) in H9c2 cells followed by replacement with ES-CM or culture medium. H9c2 cells treated with H2O2 were exposed to ES-CM, and ES-CM+cell survival protein phosphatidyl-inositol 3-kinase (PI-3k/Akt) inhibitor, LY294002 or extracellular signal-regulated kinase (ERK1/2), PD98050. After 24 hours, H9c2 cells treated with ES-CM demonstrated significant increase in cell survival. ES-CM significantly inhibited (p<0.05) apoptosis determined by TUNEL staining, apoptotic ELISA and caspase-3 activity. Importantly, enhanced cell survival and inhibited apoptosis with ES-CM was abolished with LY294002. In contrast, PD98050 shows no effect on ES-CM increased cell survival. Furthermore, H2O2 induced apoptosis is associated with decreased levels of phosphorylated (p) Akt activity. Following treatment with ES-CM, we observed a decrease in apoptosis with an increase in pAkt, and increased activity was attenuated with Akt inhibitor, suggesting that the Akt pathway is involved in the decreased apoptosis and cell survival provided by ES-CM. In contrast, we observed no change in ES-CM decreased apoptosis or pERK with PD98050. In conclusion, we suggest that ES-CM inhibited apoposis and is mediatd by Akt but not ERK pathway.
